Mosquitoes in the Culex genus are principal vectors of encephalitis viruses and lymphatic filariasis worms. Culex pipiens and C. quinquefasciatus are major vectors of West Nile virus, which is now the leading cause of mosquito-borne disease in the continental US (https://www.cdc.gov/westnile/index.html). There are concerns that climate change may cause further increases in West Nile cases. Members of the C. pipiens species complex, which includes C. quinquefasciatus, C. pipiens, C. molestus and a few other species, show different modes of evolutionary divergence and exhibit behavioral and physiological differences relevant to their vectorial capacity. Therefore, the C. pipiens complex are compelling to study from both biomedical and basic science perspectives. We have a long-standing interest in deciphering the molecular mechanisms by which the male-determining locus (M locus) controls sex-determination and other aspects of male biology in mosquitoes. We focus on C. quinquefasciatus in this proposal for both fundamental and translational considerations. As the male-determining factor (M factor) in C. quinquefasciatus is different from any known M factors in mosquitoes, new insights will be gleaned into sex-determination governed by a novel master regulator in C. quinquefasciatus. The proposed research will also lay the foundation for studies into the evolution of homomorphic sex chromosomes and provide paternally-inherited markers to facilitate investigations into the intriguing evolutionary dynamics of the C. pipiens complex. From an applied perspective, the proposed research will enable innovative applications to control C. quinquefasciatus populations through the introduction of a sex bias towards the non-biting males. By analyzing various RNAseq datasets followed by experimental verifications, we have recently identified two genes in the M locus including a strong candidate for a novel M factor we named cMf. We have also obtained a high quality male C. quinquefasciatus genome assembly by separating paternal and maternal long-read sequences and by Bionano scaffolding. We will pursue two specific aims: 1) systematically characterize the genetic contents of the M and m loci in C. quinquefasciatus; 2) investigate the function of selected M locus genes including cMf. This exploratory R21 will produce a systematic annotation of the genetic contents of the M and m loci and discover the function of the most important M locus genes in C. quinquefasciatus. It will lay the foundation for future research into 1) the mechanism of action of a novel M factor, 2) the evolution of the homomorphic sex chromosomes, 3) the functional elements of the m locus, and 4) applications based on sex ratio manipulation or linkage to the M locus.